Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105 Israel.
The National Institute for Biotechnology in the Negev (NIBN), Beer-Sheva, 84105 Israel.
J Biol Chem. 2018 Aug 17;293(33):12663-12680. doi: 10.1074/jbc.RA117.000871. Epub 2018 Jun 22.
Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPI (APPI-4M), an APPI variant with a KLK6 inhibition constant ( ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPI). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPI/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.
人组织激肽释放酶（KLK）蛋白酶是具有重要功能的激素样信号分子，在癌症病理生理学中。KLK 相关肽酶 6（KLK6），特别是，在几种类型的癌症中高度上调，其活性增加促进癌症侵袭和转移。这一特征表明 KLK6 是治疗干预的一个有吸引力的靶点。然而，尚未报道专门针对 KLK6 的抑制剂，可能是因为 KLK6 与其他丝氨酸蛋白酶具有高度的序列同源性和结构相似性，并抵抗许多多肽抑制剂的抑制。在这里，我们通过基于流式细胞术的酵母展示突变文库筛选，提出了一种创新的组合方法来设计 KLK6 抑制剂，该文库展示了人淀粉样前体蛋白 Kunitz 蛋白酶抑制剂结构域（APPI）的突变体，APPI 是其他丝氨酸蛋白酶（如阴离子和阳离子胰蛋白酶）的抑制剂。基于该筛选，我们生成了 APPI（APPI-4M），APPI 的一种变体，对 KLK6 的抑制常数（ ）为 160pm，转化时间为 10 天。据我们所知，APPI-4M 是迄今为止报道的最有效的 KLK6 抑制剂，与 WT APPI（APPI）相比，其亲和力提高了 146 倍，蛋白水解稳定性提高了 13 倍。我们进一步证明，APPI-4M 在依赖 KLK6 的乳腺癌侵袭的细胞模型中作为功能性抑制剂发挥作用。最后，APPI/KLK6 和 APPI-4M/KLK6 复合物的晶体结构揭示了 APPI-4M 改善 KLK6 结合和蛋白水解稳定性的结构和机制基础。我们预计，APPI-4M 将作为成像剂和治疗剂具有重要的转化潜力。
